Hindawi Publishing Corporation

BioMed Research International

Volume 2013, Article ID 297501, 11 pages
http://dx.doi.org/10.1155/2013/297501

Review Article
Genetic and Functional Profiling of Crohn’s Disease: Autophagy
Mechanism and Susceptibility to Infectious Diseases

Annalisa Marcuzzi,1 Anna Monica Bianco,1 Martina Girardelli,1 Alberto Tommasini,1

Stefano Martelossi,1 Lorenzo Monasta,1 and Sergio Crovella1,2
1
Institute for Maternal and Child Health—IRCCS “Burlo Garofolo” of Trieste, Via dell’Istria 65/1, 34137 Trieste, Italy
2
University of Trieste, 34127 Trieste, Italy

Correspondence should be addressed to Annalisa Marcuzzi; marcuzzi@burlo.trieste.it

Received 27 February 2013; Accepted 20 March 2013

Academic Editor: Enrique Medina-Acosta

Copyright © 2013 Annalisa Marcuzzi et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Crohn’s disease is a complex disease in which genome, microbiome, and environment interact to produce the immunological
background of the disease. Disease in childhood is more extensive and characterized by a rapid progression, leading to severe
repercussions in the course of the disorder. Several genetic variations have been associated with an increased risk of developing the
disease and most of these are also implicated in other autoimmune disorders. The gut has many tiers of defense against incursion by
luminal microbes, including the epithelial barrier and the innate and adaptive immune responses. Moreover, recent evidence shows
that bacterial and viral infections, as well as inflammasome genes and genes involved in the autophagy process, are implicated in
Crohn’s disease pathogenesis. The aim of this review is to establish how much the diagnostic system can improve, thus increasing the
success of Crohn’s disease diagnosis. The major expectation for the near future is to be able to anticipate the possible consequences
of the disease already in childhood, thus preventing associated complications, and to choose the best treatment for each patient.

1. Introduction of steroids to control the first flare [8]. However, as the

Crohn’s disease (CD) is a chronic form of inflammatory bowel
disease (IBD) that can affect any part of the gastrointestinal
tract, from the mouth to the anus. However, it most com-
monly affects the colon and terminal ileum [1] with up to
75% of patients having ileal disease with or without colonic
involvement [2]. It is a debilitating disorder with an overall
prevalence of 0.5%–1% of the general population [3, 4]. CD
differs from other types of IBDs because in patients with CD
the inflammation is often continuous and with involvement
of the mucosa [5].
Complications are common but not a constant: disease
progression is marked by severe colitis, strictures and peri-
anal fistulas, typically requiring surgery [6, 7].
Beaugerie et al. [8] recently reported three factors that at
the time of the diagnosis increase the chance of developing a
disabling disease in the following five years: (A) age <40 years,
(B) presence of perianal lesions, and (C) the requirement
median age at diagnosis is 27 years, patients may live with
CD for more than 50 years in the Western world, where life
expectancy of patients exceeds 70 years.
The age of onset is frequently in the second decade
of life, and most patients progress to a relapsing disease
characterized by abdominal pain, bloody diarrhea, vomit,
and weight loss.
Although CD normally manifests in adulthood, it can
be present in childhood before the age of 2 years [9].
The early onset Crohn’s disease (EOCD) is typically more
extensive (beyond the colon and/or oral or perianal dis-
ease) and characterized by rapid progression, leading to
severe repercussions in disease development [10]. Diagnosis
is particularly challenging in children in which presenting
symptoms may vary widely and may only consist of subtle
extraintestinal manifestations [11]. This often leads to a typical
delay in the diagnosis of pediatric IBD, ranging from 4 weeks
in severe colitis to 6-7 months in milder disease. Reducing
2 BioMed Research International
this diagnostic delay is important, since a long period of
unmanaged symptoms can significantly impact on growth
and early treatment is essential to preserve long-term quality
of life [12, 13]. Thus, a sensitive yet noninvasive tool for
the identification of patients at high risk of IBD, and there-
fore warranting endoscopic evaluation, would be a valuable
diagnostic aid. There are specific clinical, therapeutic, and
psychosocial issues specific to children with IBD that must
be considered to ensure prompt diagnosis and appropriate
medical management.
The etiopathogenesis of CD is unclear. It remains to
be determined whether this disease represents an abnormal
response to normal antigenic stimuli or an appropriate
response to persistently abnormal stimuli [14, 15]. A better
understanding of the origin of the disease and the mecha-
nisms of action is necessary to improve the prognosis of CD.
CD is a complex disorder resulting from the interaction of
genetic environmental and microbial factors. Given the diffi-
cult genotype-phenotype correlation and given the heteroge-
neous genetics, the different interactions among predisposing
factors, and not only the number of genes involved, should
be considered in order to understand the mechanisms of this
disease. Many previous and ongoing studies have sought to
identify genes and especially disease-causing variants such as
risk factors for the disease. Identification and characterization
of disease-causing variants represents one of the biggest chal-
lenges of genetics within the etiopathogenetic study of CD.
Genomewide association studies already identified several
distinct genetic polymorphisms associated with Crohn’s dis-
ease. In European individuals, NOD2 gene polymorphisms
confer by far the greatest risk for the disease. Other variations
in ATG16L1, IRGM, and IL23R genes were reported to be
highly associated with CD.
The genetic background has certainly a predisposing
role, but several alternative explanations are possible, mostly
related to lifestyle. The importance of environmental factors is
shown by an increase in the incidence rate of diseases in eth-
nic groups previously less affected, as Hispanic, Asians, and
immigrants that had moved from regions of low incidence
into areas where the incidence of the disease is higher [16].
Observation of Crohn’s patients and animal models sug-
gests a role of bacteria in the disorder [17]. Among the most
important bacteria that can adhere and invade the mucosa is
Escherichia coli [18]. The onset of the disease is quite common
after gastrointestinal infections and people suffering from this
disorder have, generally, higher concentrations of mucosal
bacteria if compared to healthy subjects [19].
A detailed picture of how genes work together and
interact with environmental and microbial factors may better
explain individual differences in CD susceptibility.
2. Etiology of CD
The complex pathophysiology of CD has, for a long time, been
an enigma [20].
Although the precise etiology of CD remains elusive,
epidemiological data conclusively indicate a deregulation of
Genetic
susceptibility
Crohn’s
disease
Infectious Immune
disease response
Figure 1: Crohn’s disease is a heterogeneous disorder of multifacto-
rial etiology in which genetic, environmental, and microbial factors,
together with the immunological response, interact to produce the
disease.
the immune response against the luminal flora in a genetically
susceptible host [21] (Figure 1).
It is commonly assumed that CD is a heterogeneous disor-
der of multifactorial etiology in which genome, microbiome
(hereditability), and environment interact to produce the
immunological background of the disease. It is probable that
patients have a genetic predisposition for the development
of the disease coupled with immunoregulation disturbances
[22].
2.1. Genetic Susceptibility: Greater Weighing Factor in Early
Onset Crohn Disease. The epidemiologic evidence of the
role of genetic factors in the pathogenesis of the dis-
ease came from studies demonstrating higher rates of CD
among individuals of Caucasian and Jewish ethnicity, familial
aggregation of CD, and higher concordance rates of both
twins developing CD in monozygotic compared to dizygotic
twins. Due to the complexity of the disease, the search for
specific CD susceptibility genes has been very difficult so
far. Despite the large number of genomewide associations
(GWAS) established to date, most complex diseases (not
monogenic) have only managed to explain some additional
percentage of the hereditability estimates. The source of this
missing hereditability is the subject of much debate with
various explanations: overestimates of original heritability
statistics, underpowered GWAS studies to detect common
variants, poorly investigated epistasis and gene-environment
interactions, and rare genetic variants [23]. In the attempt
to explain some of this missing hereditability, researchers
have adopted several complementary strategies. Combined
genotypes, “private genes,” and epigenetic markers may
account for this missing hereditability: monogenic immunity
disorders are increasingly diagnosed in patients with EOCD
[24]. Advances in bioinformatics have now made it possible
to perform GWAS using copy number variation probes. By
several GWAS and meta-analysis studies many genes have
been associated with CD: more than 90 distinct genomic loci
have been found to be associated with an increased risk of
BioMed Research International 3

Genetics
susceptibility loci Autophagy
NOD2, ATG16, IRGM,
LRRK2

New loci CD associated [23] Other Th17 pathway

ZMIZ1, FUT2, CPEB4, IL23R, IL12B, STAT3,
5q31, IKZF1, AK2, TYK2
ZPBP2TNFS18-4, FASLG,,
ORMAD2 ADAM30, IL6ST,
Early-onset associated loci [30] ILL31R, CREB5, AZF1,
RIPK2, RASGRP1, SPRED1,
LGALS9, NOS2, IFNGR2-1, Immune-mediated
CREM, C11ORF30, PTPN22, CCR6, IL2RA,
PTGER4, ORMDL3, IL18RAP,
IBD associated loci GSMDL 16p11.2, ERAP2,
ITLN1,
CCL2/CCL7,
TNFSF11, BACH2,
TAGAP, VAMP,
TNFSF15, ICOSLG

Figure 2: More than 90 distinct genomic susceptibility loci have been found to be associated with an increased risk of developing CD. The
genes variants relate largely to the innate immunity genes, in particular to the disruption of the innate and adaptative arms of the immune
systems, to the process of autophagy, to the epithelial barrier function, and to the activation of the endoplasmic reticulum stress response.

developing CD. Genes with replicated evidence for strong
association suggest that these variants relate largely to the
innate immunity genes, in particular to the disruption of
the innate and adaptive arms of the immune system, to the
process of autophagy, to the epithelial barrier function, and
to the activation of the endoplasmic reticulum stress response
[25–27] (Figure 2).
Most loci are in relation with the CD phenotype and
many loci are implicated in other immune-mediated dis-
orders, most notably with ankylosing spondylitis, erythema
nodosum, and psoriasis [28]. Several genes are involved in
primary immunodeficiencies, characterized by a dysfunc-
tional immune system resulting in severe infections [26, 29,
30]. In the last years defective processing of intracellular
bacteria has become a central theme. A considerable overlap
has also been observed between susceptibility loci for CD and
susceptibility for infectious diseases [27].
Genetic susceptibility is thought to play a more important
role in the etiology of early rather than late onset CD [31].
This is supported by a higher rate of positive family history of
CD in patients with a younger age at diagnosis with respect
to patients with older age at diagnosis, suggesting that an
earlier presentation may be due to a higher burden of disease-
causing mutations in the genomes of these affected children
compared to those in whom disease manifests later in life
[32]. EOCD presents a more aggressive phenotype; in fact
earlier age at diagnosis is associated with a greater need for
surgery [33]. EOCD candidate susceptibility genes have been
identified using linkage analysis and gene sequencing in two
unrelated consanguineous families [34]. Some gene/loci may
be specific to pediatric-onset CD and that is documented by
recent GWAS focused on a pediatric cohort highlighting the
implication of novel pathways and interaction between the
two different onsets [35, 36].
Moreover, environmental factors such as smoking are less
likely to be exerting an influence on the disease in pediatric
cohorts [37].
2.2. Gastrointestinal Microbiota: Host Genome-Microbe Inter-
actions in Crohn’s Pathogenesis. Considering epidemiolog-
ical, genetic and immunological data, it is probable that
patients have a genetic predisposition for the development of
the disease coupled with disturbances in both immunoregu-
lation, and intestinal microbiota. The disease can be triggered
by any of a number of different factors and sustained by
an abnormal immune response to these factors. Rather,
the intensive interaction between intestinal epithelial cells
and immune competent cells is critical to maintain and
perpetuate the chronic inflammatory process characteristic
of CD [22]. The genetic and pathological complexity of CD
is particularly well suited for testing whether interactively
redefining disease diagnoses can enhance the value of genetic
and pathogenetic studies. Precision in the characterization of
the disease would make defining the impact of host-gene-
microbial interactions on the disease process more robust.
The human body is inhabited by a vast number of
bacteria, archaea, viruses, and unicellular eukaryotes. The
microbiota represents a collection of microorganisms that
live in peaceful coexistence with their hosts [38]. By far,
the most heavily colonized organ is the gastrointestinal tract
(GIT) and the colon alone is estimated to contain over
70% of all the microbes [39] and represents a major surface
4 BioMed Research International
susceptibility
Healthy gut
PAMPs Lumen
Mucus layer
Epithelial barrier
Lamina propria
Physiological inflammation
Homeostasis
Commensal Autoinflammatory
bacteria bacteria
Figure 3: The role of microbiota in Crohn’s disease pathogenesis. The interplay between the host microbiota and the environmental factors
in a genetic susceptible host results in a progressive inflammatory damage to the host intestinal mucosa.
for microbial colonization. It is estimated that the human
microbiota, not homogeneous in the GIT, contains as many as
1014 bacterial cells [40, 41] and the number of bacterial species
present in the human gut is estimated to be 500 to 1000 [42].
Nevertheless, a recent analysis involving multiple subjects
has suggested that the collective human gut microbiota is
composed of over 35000 bacterial species [43].
Colonization of the human gut with microbes begins
immediately at birth; in fact infants are exposed to a complex
microbial population upon the passage through the birth
canal [44] and it is known that infants delivered through
cesarean section have different microbial compositions com-
pared to vaginally delivered infants [45]. It has been shown
that the microbiota of adult monozygotic and dizygotic twins
was equally similar to that of their siblings, suggesting that
the colonization by the microbiota from a shared mother
was more decisive in determining their adult microbiota than
their genetic makeup [46] (Figure 3).
Several studies have shown that host genetics can impact
the microbial composition of the gut [47, 48]. The central role
of gut microbiota in the development of mucosal immunity
is not surprising considering that the intestinal mucosa
represents the largest surface area in contact with the anti-
gens of the external environment called PAMPs (pathogen-
associated molecular patterns). Additionally, the dense car-
pet of the gut microbiota overlying the mucosa normally
accounts for the largest proportion of the antigens presented
to the resident immune cells and those stimulating the pattern
recognition receptors such as the NOD-like receptors (NLRs)
of the intestinal epithelial cells [49]. The GI (gastrointestinal
gut) microbiome of healthy humans is dominated by four
Genetic
Environmental
factors
Pathological gut
Defensins
IgA, IgG
Damaged
epithelial
barrier
Chronic inflammation
Mucolytic Harmful
bacteria bacteria
major bacterial phyla: Firmicutes, Bacteroidetes, and to a
lesser degree Proteobacteria and Actinobacteria [50]. Many
studies have observed imbalances or dysbioses in the GI
microbiomes of CD patients [51, 52]. In CD patients biodiver-
sity is decreased, with a lower proportion of Firmicutes and an
increase in Gammaproteobacteria [53]. In CD, proportions of
the Clostridia are altered: the Roseburia and Faecalibacterium
genera of the Lachnospiraceae and Ruminococcaceae families
are decreased, whereas Ruminococcus gnavus increases [54].
An important concept in the pathogenesis of CD is that
bacterial and viral interactions occur in a host gene-specific
manner [55, 56]. Surgical diversion of the fecal stream
ameliorates inflammation. In addition, the evaluation of the
microbial populations in surgically resected tissue samples
of small bowel and colon from CD patients and non-CD
controls, by rRNA sequence analysis, showed that specific
flora was not enriched in small bowel or colon from CD
patients. However, a subset of CD samples showed alterations
in the representations of the Bacteroides and Firmicutes
[43, 57]. Moreover, several studies have shown that the gut
microbiota is altered in IBD patients. For example, biopsy
samples from CD patients were used to prepare bacterial
DNA which was amplified using universal bacterial 16S rRNA
primers [58], and a significant increase in Proteobacteria and
Bacteroidetes was found in CD patients compared to controls,
with a decrease in Clostridia. Metagenomic approaches were
used to analyze fecal samples from Crohn’s patients and
healthy donors and revealed reduced complexity of the
Firmicutes in affected individuals [59]. Finally, the evidence
that intestinal bacteria play an important role in CD patients
is that antibiotics help some patients and can ameliorate
BioMed Research International
IL-17A
IL-17F
IL-21
IL-22 IFN-𝛾
IL-26 TNF-𝛼
IL-6 Th17 IL-6
TNF-𝛼
Th1
Proinflammatory cytokines
Figure 4: Up- and Downregulation of the proinflammatory cytokines evidenced in the immune system dysregulation of CD patients.
disease activity. Moreover metronidazole is an important
therapeutic agent for certain complications of CD such as
fistulising disease. Viral infection is required to generate the
Paneth cell defect found in ATG16L1 mice [60], suggesting
that in addition to human bacterial microbiota, viral or fungal
commensals may play a role in CD pathogenesis.
2.3. Immunological Response in CD
2.3.1. Th1 and Th17 Implicated in CD Pathogenesis. Although
the exact CD etiology is still not completely understood,
several studies indicate that its pathogenesis is characterized
by an exaggerated immune response in genetically susceptible
individuals.
CD patients suffer from marked immune system dereg-
ulation. The inflammation seen in these patients is charac-
terized by pronounced Th1 and Th17 responses [61] involving
upregulation of proinflammatory cytokines IL-1, IL-6, IL-12,
TNF-𝛼, IFN-𝛾, IL-23, and IL-17 and downregulation of IL- 10,
but it is not clear whether this is a cause or a consequence of
the disease [62] (Figure 4).
Th1 cells are commonly assumed to be associated with CD
development and produce IFN- 𝛾, and their primary role is
the protection against intracellular microbes. IFN-𝛾 secreting
lamina propria lymphocytes are abundant in the mucosa of
CD patients: this condition is marked at CD onset (mucosal
T cells appear to mount a typical Th1 response that resembles
an acute infectious process) and disappears in late CD.
Recently, several studies showed the pivotal role of the
imbalance of regulatory T cells (Treg) and Th17 in CD. Treg
cells are important for the control of the immune response to
self-antigens preventing autoimmunity and maintaining self-
tolerance [63]. In contrast, IL-17 producing Th17 cells were
recognized as a novel group of T cells which play a major
role in autoimmunity. The gastrointestinal immune system
has to maintain both a state of tolerance toward intestinal
antigens and the ability to combat pathogens. In CD this
balance is lost and the effects of proinflammatory T cells
outnumber the tolerizing, anti-inflammatory effects of Treg
5
IL-10
TGF-𝛽
Treg
Anti-inflammatory cytokines
cells. The discovery that Th17 cells, which express the IL-
23 receptor (IL-23R), play a role in CD pathogenesis was
supported by recent GWAS studies demonstrating that IL-
23R and other genes involved in the differentiation of Th17
cells are susceptibility genes.
To confirm the link between immune response and
genetic susceptibility in the pathogenesis of CD there are
several recent lines of evidence that the key role is played
by autophagy that includes the antigen presentation and the
production of proinflammatory cytokines. The relationship
between autophagy and microbes, indeed, has remained ill-
defined until a recent convergence of studies showing that
autophagy is an innate immune defense against bacteria, pro-
tozoa, and viral pathogens [64]. It is commonly assumed that
the role of autophagy in addition to eliminating intracellular
pathogens [65] contributes to MHC II restricted endogenous
antigen presentation. It is an effector of Th1/Th2 polarization,
affects B and T cell homeostasis and repertoire selection,
delivers cytosolic PAMP or danger associated molecular
patterns to endosomal toll-like receptors (TLR), and acts
as an innate immunity effector downstream of TLR [66].
Polymorphisms in autophagy genes result in deregulation of
these processes and affect gut homeostasis: genetic variants of
autophagy genes have been linked to CD.
2.3.2. The Role of the NLRP3 Inflammasome in the Patho-
genesis of CD. Inflammasomes are cytoplasmic multipro-
tein complexes that function as sensors of endogenous
or exogenous PAMPs. They are composed of one of sev-
eral nucleotide-binding oligomerization-domain protein-like
receptors (NLRs), including NLRP1, NLRP3, NLRP6, and
NLRPC4. Upon sensing the relevant signal, they assemble,
typically together with an adaptor protein, an apoptosis-
associated speck-like protein (ASC) or a caspase activating
and recruitment domain 8 (CARD8), into a multiprotein
complex that governs caspase-1 activation and subsequent
cleavage of effector proinflammatory cytokines including
pro-IL-1𝛽 and pro-IL-18.
6 BioMed Research International
Recently several studies highlighted with particular
emphasis the relevance and the role of NLRP3 (previously
known as CIAS1 and NLRP3) in the pathogenesis of CD
[67, 68].
There is evidence suggesting that NLRP3 is able to
respond to a variety of signals: adenosine triphosphate
(ATP), nigericin, maitotoxin, Staphylococcus aureus and Lis-
teria monocytogenes [69], and RNA and uric acid crystals
(monosodium urate and calcium pyrophosphate dehydrate)
released from dying cells [70, 71].
Literature data showed that the proinflammatory com-
pound muramyl-dipeptide, the minimal bioactive peptido-
glycan motif common to all bacteria, was an activator of the
NLRP3 inflammasome, which suggested a very interesting
connection between NOD2 and NALP3 [72].
On the other hand, Kanneganti et al. suggested that
bacterial RNA and small antiviral compounds are the specific
ligands of NLRP3 rather than MDP [70].
Anyway, NLRP3 plays a pivotal role in the inflammation
regulating the activation of the caspase-1 and processing of
IL-1𝛽, two key mediators involved in the pathogenesis of the
more common inflammatory disorders [73].
More recently, in an increasingly complicated picture,
Elinav et al. described a novel regulatory sensing system in
the colon, dependent on the NLRP6 inflammasome [74], and
von Kampen et al. showed that CARD8 negatively regulates
NOD-2 mediated signaling [75]. These current data further
underline the link between the different components of the
CD etiopathogenesis that are strongly correlated.
3. Autophagy in CD
Genomewide association studies and genetic analyses have
emphasized the involvement of autophagy processes in the
pathogenesis of inflammatory bowel diseases implicating
three component genes in CD pathogenesis: ATG16L1 [76],
IRGM [77], and NOD2 [78, 79]. These genes encode proteins
critical for autophagy, a process that mediates degradation
of intracellular proteins via vesicle-mediated delivery to the
lysosome [80, 81]. Autophagy is involved in intracellular
homeostasis, contributing to the degradation and recycling
of cytosolic contents and organelles, as well as to resistance
against infection and the removal of intracellular microbes. It
is a major degradative pathway of the cell with several critical
functions in innate and adaptive immunity [82] (Figure 5).
The ATG16L1 deficiency mouse showed Paneth cell dys-
function with aberrant exocytosis, as well as an altered
transcriptional profile, characterized by increased expression
of pro-inflammatory cytokines and lipid metabolism genes
like Paneth cell phenotype of CD patients [83]. Nevertheless
an important observation derived from the ATG16L1 mouse
model was that the murine norovirus infection, as well as
the presence of the commensal bacteria, was required for
the generation of these specific Paneth cell abnormalities
[84]. ATG16L1 is essential for all forms of autophagy, and the
coding mutation T300A is associated with increased risk of
CD. Despite its ubiquitous expression, the defects associated
with ATG16L1 polymorphisms have so far been described
only within the gut, probably owing to the high microbial load
in this tissue.
Nucleotide-binding-oligomerization-domain- (NOD-)
like receptors (NLRs) represent ancient sentinels of the host
innate immune system, and genetic variants in NLR genes
are associated with complex chronic inflammatory barrier
diseases [85]. The NOD2 gene is an intracellular sensor for
the bacterial cell wall component muramyl-dipeptide, and
loss-of-function variants in the human NOD2 gene have
been associated with an increased susceptibility for CD
[86, 87] in Caucasian populations of European ancestry [88],
and particularly for ileal disease [89], and were found to be
an important regulator of the commensal gut microbiota in
mice [90]. NOD2 recognizes components of the bacterial cell
wall and elicits an NF-𝜅B response and mediates the release
of defensins, which are antimicrobial peptides. Evidence
from MDP stimulation of NOD2-activated autophagy shows
a link between genetic risk loci and highlights the importance
of defining disease associated pathways and the potential of
new roles for known genes [78]. Epithelial cells and dendritic
cells containing Crohn’s-disease-associated ATG16L1 and
NOD2 variants show defects in antibacterial autophagy
[79, 91]. In dendritic cells, these defects are associated with
an impaired ability to present exogenous antigens to CD4+T
cells [78]. A discussed model of Crohn’s disease is the one in
which individuals are genetically susceptible to a pathogen
that triggers a compensatory and harmful immune response.
Antibacterial autophagy, through ATG16L1, NOD2, and
potentially other genes (IRGM), is consistent with this
model. However, one of the most important experimental
supports for this model comes from an unrelated study using
Citrobacter rodentium to induce intestinal inflammation
in NOD2 –/– mice [92]. These results illustrate a close
relationship between NOD2, ATG16L1, and autophagy,
affecting intracellular processing and communication
with the adaptive immune system suggesting that genetic
polymorphisms may affect both pathways concomitantly.
IRGM belongs to the p47 Immunity-Related GTPase
(IRG) family and is linked to CD by GWAS as a protein
that is implicated in the autophagy mechanism [93]. The
analysis of the interactions between 44 autophagy-associated
human proteins and 83 viral proteins belonging to different
RNA virus families revealed that IRGM was the autophagy-
associated protein most targeted by these viruses. IRGM can
interact with 12 viral proteins belonging to different viruses,
such as HCV and HIV-1 [94, 95]. A recent study suggests
that a polymorphism in IRGM could affect the binding and
the consequent misregulation by a specific miRNA (miR-196)
that is highly expressed in the intestinal tissue of patients with
Crohn’s disease. The consequence is that the xenophagy flux is
not well regulated leading to the accumulation of bacteria in
the lysosomal compartment. This study showed that greater
IRGM expression leads to both colocalization of adherent
invasive E. coli (AIEC) with the autophagy machinery and
increased intracellular survival of the bacteria [96]. This and
other strains of E. coli are more abundant in the mucosa of
CD patients [90]. The ability of IRGM to induce autophagy
and limit the replication of intracellular bacteria has been
demonstrated with mycobacteria by inducing mitochondrial
BioMed Research International 7

MDP
Bacteria

CARD1 CARD 2 NOD LRR

Plasma NOD 2
membrane

CARD1 CARD 2 NOD LRR

CARD1 CARD 2 NOD LRR
(b)
ATG16 WD repeat ATG16 WD repeat
ATG16L1 ATG12
ATG5

(c) IRGM IIGP Endosome

Amphisome
Autolysosome Phagophore

Autophagosome

Lysosome

Degradation Maturation Closure Elongation Initiation

(a)
Autophagy process

Figure 5: CD pathogenesis and autophagy: susceptible CD genes ATG16L1, NOD2, and IRGM are proteins critical for the autophagy
process. (a) The process of mammalian autophagy is divided into the following principal steps: initiation, elongation, closure, maturation,
and degradation. (b) At the bacterial entry site NOD2 activated by MDP recruit ATG16L1 to the plasma membrane. Follow the assembling
of the ATG5-ATG12 complex, stabilized by ATG16L1, that facilitates the formation of an autophagosome around the invading bacterium. (c)
IRGM, another autophagy-related gene, could be involved in the final steps of the degradation step.

depolarization and can increase ROS production and cell
death [97]. Finally IRGM could regulate inflammation by
either regulating intracellular pathogens or cellular home-
ostasis much like ATG16L1.
These data provide further information and support for
the hypothesis that microbial/viral interactions with the
intestinal mucosa are required for disease generation and
suggest that combinatorial models for CD pathogenesis are
most relevant for the study of human disease pathogenesis.
4. Concluding Remarks
The diagnosis of CD is reached through the results of clinical,
laboratory, radiographic, endoscopic, and histologic analyses.
Radiological and endoscopic techniques are essential for
the diagnosis of CD since its onset and are useful in assessing
the inflammatory status of the intestinal mucosa. However,
endoscopy is an invasive procedure. In children it can be
traumatic and could have critical implications due to the
more severe clinical manifestation and complication of the
pediatric disease, that make the intestinal mucosal extremely
thin and at risk of perforation.
Noninvasive tests for CD already exist, including antibod-
ies, imaging-based screens, and fecal biomarkers [98]. The
specificity of existing methods ranges from 89% to 95% for
CD and other inflammatory bowel diseases. However, these
methods are limited to active disease and poorly sensitive
(∼55%). Their outcome can be confounded by other diseases,
further limiting their clinical utility. Recently, high expecta-
tions are placed in diagnostic studies of the gastrointestinal
microbiota, but further validations will be necessary before
this tool is accepted in clinical practice [32, 99].
Research is moving forward in order to identify new and
valid biomarkers for the diagnosis of the disease with the aim
of replacing the use of invasive techniques.
Currently, only the measurement of fecal calprotectin
levels has achieved a place in clinical routine practice and is
used as a marker for noninvasive determination of intestinal
8 BioMed Research International
inflammation [99, 100]. This protein is an ideal marker
because it is not degraded by the human microbiota. Levels of
fecal calprotectin significantly increase in patients with CD,
ulcerative colitis, infectious colitis, and, to a lesser extent,
in tumors of the colon rectum, but not in patients with
functional disorders, as in the case of the irritable bowel
syndrome. In CD, the calprotectin assay reflects the activity
of the disease, monitoring its progression, and can contribute
to the decision about the correct medication strategy.
The application of this test in the pediatric population
is a good result. Although the test is not yet able to replace
diagnostic colonoscopy, it can be a good indicator for the
decision to use or delay the use of invasive investigations [99].
Recently, Vitali et al. suggested the use of high-mobility
group box1 (HMGB1) as a novel marker of intestinal mucosal
inflammation. HMGB1 is today regarded as a pleiotropic
cytokine, that is passively released by necrotic cells, but not
from apoptotic cells. Moreover HMGB1 could be actively
secreted from some types of immune cells in response
to lipopolysaccharide (LPS), IFN-𝛾, and TNF-𝛼. There is
evidence that HMGB1 is secreted in the stools of these
patients and not detectable in controls [101].
The relationship between the genetic susceptibility and
the microbiome could be considered in the disease diagnosis.
There are several international human microbiome projects
that have focused initially on the bacterial component of the
microbiome. The evidence that bacteria play an important
role in CD includes the observation that surgical diversion
of the fecal stream ameliorates the inflammation and that
antibiotics help some patients. Moreover other evidence is
showed in mouse models of colitis where virus, bacteria,
or both acting together can contribute to the pathology via
signaling through innate immune sensors and regulation of
pro- and anti-inflammatory cytokines [82]. The microbiome
varies from person to person and such variation could pro-
vide environmental inputs that contribute to the incidence of
CD, within the genetic foundation revealed by GWAS [102].
The concept of dysbiosis as a contributor to CD is correlated
with intestinal bacteria communities, so the changes in the
bacterial microbiota could have a potential role in the disease.
However, this hypothesis needs to be expanded to include
specific interactions between individual bacteria and host
genes.
Moreover, another ambitious goal is the identification
of a genetic pattern able to associate specific phenotypic
characteristics to CD patients or to anticipate the possible
consequences of the disease already in childhood and thus
prevent complications associated with the disease and to
choose the best treatment for each patient.
A rapid diagnosis is fundamental to avoid a growth
delay or complications of the disease typical of the pediatric
disease leading to surgery. In some cases, genetic studies have
provided useful information for the identification of specific
mutations that predict risk of stenosis and surgery and/or
disease localization in pediatric-onset CD [103, 104].
The difficulty of finding a common genetic pattern of
association is caused by the multifactorial feature of the
disease that shows different characterizations by world region
and race.
In conclusion, it would certainly be useful to be able
to create a biological algorithm that helps clinicians in the
identification and classification of the disease and to deter-
mine the pharmacological care. This algorithm could include
not only the known and principal factors predisposing to
the disease, but also the gene-microbiome interaction and
could help identify novel markers in patients with familiar
history of EOCD. This could represent a major advance for
early-onset diagnosis as specific tests might be developed to
improve counselling, while direct identification of modifier
genes might assist in the recognition of new genetic, environ-
mental, and microbial causes of CD.
Conflict of Interests
The authors declare they have no conflict of interests related
to the issues discussed in this paper.
Authors’ Contribution
Annalisa Marcuzzi and Anna Monica Bianco contributed
equally to this study.
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